Menisci are fibrocartilaginous tissues situated between the femoral condyles and the tibial plateau that are essential for normal biomechanical function of the knee. Meniscal injuries are a significant clinical problem as each year 850,000 meniscal surgeries occur in the United States and nearly twice as many worldwide. Tears, particularly in the avascular inner zone, do not heal well even with suturing or conservative treatments and frequently require menisectomy, which ultimately leads to the development of osteoarthritis (OA). Increased levels of inflammatory cytokines, such as interleukin-1 (IL-1), have been measured in injured and degenerative joints. In addition, we have shown that IL-1 significantly decreases repair strength, cell proliferation, and tissue formation at the interface of an in vitro meniscal repair model system. Tissue engineering strategies that use a combination of cells, biomaterials, and biologically active molecules to promote tissue repair have shown some success but fail under inflammatory conditions. Therefore, the novel combination of gene therapy and tissue engineering in this application could harness the power of the reparative cells to deliver anti-inflammatory drugs during the repair process. Our primary goal is to develop combined tissue engineering and gene therapy treatments that will promote meniscal repair in the pro-inflammatory environment after meniscal injury. The central hypothesis for this project is that viral mediated drug delivery of IL-1 receptor antagonist (IL-1ra) from reparative cells in biological matrix will promote integrative meniscal repair following injury. The primary goal will be accomplished by testing the central hypothesis with the following specific aims: Specific Aim 1: Develop a cell and matrix based tissue engineering treatment to promote in vitro integrative meniscal repair. We will use our novel in vitro model of integrative repair of the meniscus. In this model, two concentric and adjacent meniscal explants naturally exhibit interfacial repair over several weeks of culture in vitro. We will fill the repair interface with various acellular biological matrices, including type I collagen or fibrin. In addition, mesenchyma stem cells (MSCs) or chondrogenically differentiated MSCs will be seeded in the matrices and delivered to the repair interface. Meniscal repair will be assessed over 28 days in vitro by measuring the integrative shear strength of repair, visualization of the repair interface with safranin O and fast green histological staining and immunostaining for types I and II collagen and aggrecan. Microscopy will also be utilized to assess collagen structure and organization in the repair interface. Specific Aim 2: Develop a combined gene therapy and tissue engineering treatment to promote in vitro integrative meniscal repair in the presence of IL-1. Using our in vitro model of integrative repair of the meniscus, we will directly transduce the injured meniscal tissue with lentivirus to deliver constitutively active or inducible enhanced green fluorescent protein (eGFP) or IL-1ra. As an alternative, we will transduce MSCs with the constitutive or inducible eGFP or IL-1ra and seed these cells in the repair interface. Non-transduced tissue or MSCs will serve as a control. In the presence of IL-1?, we will assess meniscal healing over 28 days by measuring shear strength of repair, matrix metalloproteinase (MMP) activity, nitric oxide (NO) and glycosaminoglycan (GAG) release, collagen degradation, and histology. IL-1ra production will be measured by ELISA and confocal microscopy will allow visualization of the eGFP transduced cells throughout culture. Specific Aim 3: Evaluate the combined gene therapy and tissue engineering treatment in an in vivo longitudinal meniscal tear model. Using a porcine longitudinal tear model in the avascular inner region of the meniscus, we will assess meniscal repair in response to IL-1ra overexpression via lentiviral gene therapy. Meniscal tears will be treated with the combined gene therapy and tissue engineering treatment that resulted in the largest shear strength of repair in Specific Aim 2, the combined treatment expressing eGFP in lieu of IL-1ra as a control, or untreated. After 3 and 7 days confocal microscopy will be performed to track eGFP transduced cells. In the remaining animals, serum will be collected over time and twelve weeks post-operatively meniscal healing, osteoarthritis severity, and synovitis will be assessed by gross examination and histological grading. Serum and synovial fluid will be collected to assess biomarkers, including IL-1?, IL-1?, IL-1ra, TNF-?, IL-4, IL- 10, MMP activity, and collagen degradation. The long-term goals of this study are to develop strategies to promote meniscal repair following joint injury and subsequently protect against the development of OA. Potential novel gene therapy and tissue engineering treatments identified in these studies could be translated to subsequent clinical trials in patients. Relevance of the proposed work to United States public health and the VA patient care mission. Meniscal injuries are highly relevant to the military and to the VA Health System. More than 100,000 meniscal injuries occurred in military personnel during the 1998-2006 period. Many meniscal injuries are treated by meniscectomies, and this procedure is almost invariably followed by osteoarthritis-degenerative arthritis in that joint, with resultant severe disability. Current methods of meniscal repair are clearly inadequate, and we need better approaches to this. Our studies of combined gene therapy and tissue engineering will lead to improvements in meniscal injury repair. They should result in better and faster recovery from meniscal injury and lead to a long-term decrease in secondary osteoarthritis for both active duty military personnel and military veterans.